Human neurokinin-3 receptor

ABSTRACT

A recombinant human neurokinin-3 receptor (hereinafter identified as human NK3R) is disclosed which has been prepared by polymerase chain reaction techniques. Also disclosed is the complete sequence of human NK3R complementary DNA; expression systems, including a CHO (chinese hamster ovarian cell line) stable expression system; and an assay using the CHO expression system. Human NK3R can be used in an assay to identify and evaluate entities that bind to the neurokinin-3 receptor.

This is a continuation of application Ser. No. 07/851,974 filed on Mar.16, 1992, now abandoned.

BACKGROUND OF THE INVENTION

The present invention concerns a cloned human neurokinin-3 receptor(hereinafter identified as human NK3R).

Neurokinin B (NKB) is a naturally occuring peptide belonging to theneurokinin family of peptides, which also includes substance P (SP) andsubstance K (SK). NKB binds preferentially to the neurokinin-3 receptor(NK3R), although it also recognizes the other two receptor subtypes (NK1and NK2) with lower affinity. As is well known in the art, neurokinin Band other tachykinins have been implicated in the pathophysiology ofnumerous diseases. Neurokinin peptides are reportedly involved innociception and neurogenic inflammation. The physiological function ofNK3R has been implicated in the regulation of enkephalin release, whilethe NK1 and NK2 receptor subtypes are involved in synaptic transmission(Laneuville et al., Life Sci., 42:1295-1305 (1988)). Since the NKBgenomic structure and subcellular distribution are different from thoseof SP and SK, the physiological function and regulatory mechanism of NKBmay be different from SP and SK.

More specifically, neurokinin B is a pharmacologically-activeneuropeptide that is produced in mammals and possesses a characteristicamino acid sequence that is illustrated below:

Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2.

Several groups have reported the cloning of certain neurokininreceptors. T. M. Fong, et al., Mol. Pharmacol., 41:24-30 (1991) havereported cloned human neurokinin-l and neurokinin-1 short form receptor.J. Yokota, et al., J. Biol. Chem., 264:17649 (1989) have reported clonedrat neurokinin-1 receptor. N. P. Gerard, et al., J. Biol. Chem.,265:20455 (1990), have reported human neurokinin-2 receptor. Cloned ratand bovine neurokinin-2 receptor have likewise been reported. Seerespectively, Y. Sasi, and S. Nakanishi, Biochem Biophys. Res. Comm.,165:695 (1989), and Y. Masu, et al., Nature 329:836 (1987). Cloned ratneurokinin-3 receptor has been reported by R. Shigemoto, et al., J.Biol. Chem., 265:623 (1990). The above references, however, neitherdisclose nor suggest the present invention.

The instant invention also concerns an assay protocol which can be usedto determine the activity in body fluids of substances that bind humanNK3R; these include neurokinin B. The assay can also be used foridentifying and evaluating substances that bind NK3R. Thus, the assaycan be used to identify neurokinin B antagonists and evaluate theirbinding affinity. Another method for an assay includes that described byM .A. Cascieri, et al., J. Biol. Chem., 258:5158 (1983). See also, forexample, R. M. Snider, et al., Science, 251:435 (1991) and S. McLean, etal., Science, 251:437 (1991). See also WIPO Patent PublicationsWO90/05525 and WO90/05729, published May 31, 1990. Methods to date haveproven inferior, in part, for failure of the animal receptor (animalNK1R, NK2R or NK3R) activity to accurately reflect that of the humanneurokinin-3 receptor. Furthermore, prior to this disclosure, human NK3Rhas not been available in a highly purified form or in substantialisolation from NK1R and/or NK2R. Use of such neurokinin receptor sourcescan not accurately depict the affinity of an agonist or an antagonistfor a human NK3R.

SUMMARY OF THE INVENTION

A novel recombinant human neurokinin-3 receptor (hereinafter identifiedas human NK3R) is disclosed which has been prepared by polymerase chainreaction techniques. Also disclosed is the complete sequence of humanNK3R complementary DNA; expression systems, including a CHO (chinesehamster ovarian cell line) stable expression system; and an assay usingthe CHO expression system.

Human NK3R can be used in an assay to identify and evaluate entitiesthat bind neurokinin B receptor or NK3R. The assay can also be used inconjunction with diagnosis and therapy to determine the body fluidconcentration of neurokinin-B related substances in patients. Inaddition, the complete sequence of the human NK3R is useful in theprocess of developing novel NK3 agonists and antagonists by computermodeling.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention concerns human neurokinin-3 receptor,said receptor being free of other human receptor proteins.

In one class this first embodiment concerns human neurokinin-3 receptor,said receptor being free of other human proteins.

Within this class, this first embodiment concerns human neurokinin-3receptor from human cells such as glioblastoma, said receptor being freeof other human proteins.

Also within this class, this first embodiment concerns humanneurokinin-3 receptor, the receptor being recombinantly produced fromnon-human cells.

In a second class, this first embodiment concerns a proteincorresponding to the amino acid sequence of human neurokinin-3 receptor,the protein comprising 465 amino acids. Within the second class thisfirst embodiment concerns a protein comprising the following 465 aminoacid sequence (SEQ ID NO:1:) depicted from the amino to the carboxyterminus:

Met Ala Thr Leu Pro Ala Ala Glu Thr Trp Ile Asp1               5                   10 Gly Gly Gly Gly Val Gly Ala AspAla Val Asn Leu         15                    20 Thr Ala Ser Leu Ala AlaGly Ala Ala Thr Gly Ala 25                  30                  35 ValGlu Thr Gly Trp Leu Gln Leu Leu Asp Gln Ala            40                  45 Gly Asn Leu Ser Ser Ser Pro Ser AlaLeu Gly Leu     50                  55                  60 Pro Val AlaSer Pro Ala Pro Ser Gln Pro Trp Ala                65                  70 Asn Leu Thr Asn Gln Phe Val GlnPro Ser Trp Arg         75                  80 Ile Ala Leu Trp Ser LeuAla Tyr Gly Val Val Val 85                  90                  95 AlaVal Ala Val Leu Gly Asn Leu Ile Val Ile Trp            100                 105 Ile Ile Leu Ala His Lys Arg Met ArgThr Val Thr     110                 115                 120 Asn Tyr PheLeu Val Asn Leu Ala Phe Ser Asp Ala                125                 130 Ser Met Ala Ala Phe Asn Thr LeuVal Asn Phe Ile         135                 140 Tyr Ala Leu His Ser GluTrp Tyr Phe Gly Ala Asn 145                 150                 155 TyrCys Arg Phe Gln Asn Phe Phe Pro Ile Thr Ala            160                 165 Val Phe Ala Ser Ile Tyr Ser Met ThrAla Ile Ala     170                 175                 180 Val Asp ArgTyr Met Ala Ile Ile Asp Pro Leu Lys                  185                  190 Pro Arg Leu Ser Ala Thr AlaThr Lys Ile Val Ile         195                 200 Gly Ser Ile Trp IleLeu Ala Phe Leu Leu Ala Phe 205                 210                 215Pro Gln Cys Leu Tyr Ser Lys Thr Lys Val Met Pro            220                 225 Gly Arg Thr Leu Cys Phe Val Gln TrpPro Glu Gly     230                 235                 240 Pro Lys GlnHis Phe Thr Tyr His Ile Ile Val Ile                 245                  250 Ile Leu Val Tyr Cys Phe ProLeu Leu Ile Met Gly         255                 260 Ile Thr Tyr Thr IleVal Gly Ile Thr Leu Trp Gly 265                 270                 275Gly Glu Ile Pro Gly Asp Thr Cys Asp Lys Tyr His        280                 285 Glu Gln Leu Lys Ala Lys Arg Lys Val ValLys Met     290                 295 Met Ile Ile Val Val Met Thr Phe AlaIle Cys Trp 300                 305                 310 Leu Pro Tyr HisIle Tyr Phe Ile Leu Thr Ala Ile         315                  320 Tyr GlnGln Leu Asn Arg Trp Lys Tyr Ile Gln Gln325                 330                 335 Val Tyr Leu Ala Ser Phe TrpLeu Ala Met Ser Ser            10 340                  345 Thr Met TyrAsn Pro Ile Ile Tyr Cys Cys Leu Asn    350                 355                 360 Lys Arg Phe Arg Ala GlyPhe Lys Arg Ala Phe Arg                 365                  370 Trp CysPro Phe Ile Lys Val Ser Ser Tyr Asp Glu         375                  380Leu Glu Leu Lys Thr Thr Arg Phe His Pro Asn Arg385                 390                 395 Gln Ser Ser Met Tyr Thr ValThr Arg Met Glu Ser             400                  405 Met Thr Val ValPhe Asp Pro Asn Asp Ala Asp Thr    410                 415                 420 Thr Arg Ser Ser Arg LysLys Arg Ala Thr Pro Arg                 425                  430 Asp ProSer Phe Asn Gly Cys Ser Arg Arg Asn Ser         435                  400Lys Ser Ala Ser Ala Thr Ser Ser Phe Ile Ser Ser445                 450                 455 Pro Tyr Thr Ser Val Asp GluTyr Ser             460                  465.

Within the second class this first embodiment also concerns a proteincomprising the foregoing amino acid sequence (SEQ ID:NO:1:), the proteinbeing free of other human receptor proteins.

A second embodiment concerns a DNA sequence encoding the humanneurokinin-3 receptor, the DNA sequence being free of other human DNAsequences.

As will be appreciated by those of skill in the art, there is asubstantial amount of redundancy in the set of codons which translatespecific amino acids. Accordingly, the invention also includesalternative base sequences wherein a codon (or codons) are replaced withanother codon, such that the amino acid sequence translated by the DNAsequence remains unchanged. For purposes of this specification, asequence bearing one or more such replaced codons will be defined as adegenerate variation. Also included are mutations (exchange ofindividual amino acids) which one of skill in the art would expect tohave no effect on functionality, such as valine for leucine, argininefor lysine and asparagine for glutamine.

One class of the second embodiment of the invention concerns thefollowing nucleotide sequence (SEQ ID NO:2:) of complementary DNAdepicted from the 5′ to the 3′ terminus:

CTATTGCAGT ATCTTTCAGC TTCCAGTCTT ATCTGAAGAC CCCGGCACCA AAGTGACCAG 60GAGGCAGAGA AGAACTTCAG AGGAGTCTCG TCTTGGGCTG CCCGTGGGTG AGTGGGAGGG 120TCCGGGACTG CAGACCGGTG GCGATGGCCA CTCTCCCAGC AGCAGAAACC TGGATAGACG 180GGGGTGGAGG CGTGGGTGCA GACGCCGTGA ACCTGACCGC CTCGCTAGCT GCCGGGGCGG 240CCACGGGGGC AGTTGAGACT GGGTGGCTGC AACTGCTGGA CCAAGCTGGC AACCTCTCCT 300CCTCCCCTTC CGCGCTGGGA CTGCCTGTGG CTTCCCCCGC GCCCTCCCAG CCCTGGGCCA 360ACCTCACCAA CCAGTTCGTG CAGCCGTCCT GGCGCATCGC GCTCTGGTCC CTGGCGTATG 420GTGTGGTGGT GGCAGTGGCA GTTTTGGGAA ATCTCATCGT CATCTGGATC ATCCTGGCCC 480ACAAGCGCAT GAGGACTGTC ACCAACTACT TCCTTGTGAA CCTGGCTTTC TCCGACGCCT 540CCATGGCCGC CTTCAACACG TTGGTCAATT TCATCTACGC GCTTCATAGC GAGTGGTACT 600TTGGCGCCAA CTACTGCCGC TTCCAGAACT TCTTTCCTAT CACAGCTGTG TTCGCCAGCA 660TCTACTGCAT GACGGCCATT GCGGTGGACA GGTATATGGC TATTATTGAT CCCTTGAAAC 720CCAGACTGTC TGCTACAGCA ACCAAGATTG TCATTGGAAG TATTTGGATT CTAGCATTTC 780TACTTGCCTT CCCTCAGTGT CTTTATTCCA AAACGAAAGT CATGCCAGGC CGTACTCTCT 840GCTTTGTGCA ATGGCCAGAA GGTCCCAAAC AACATTTCAC TTACCATATT ATCGTCATTA 900TACTGGTGTA CTGTTTCCCA TTGCTCATCA TGGGTATTAC ATACACCATT 950 GTTGGAATTACTCTCTGGGG AGGAGAAATC CCAGGAGATA CCTGTGACAA GTATCATGAG 1010 CAGCTAAAGGCCAAAAGAAA GGTTGTCAAA ATGATGATTA TTGTTGTCAT GACATTTGCT 1070 ATCTGCTGGCTGCCCTATCA TATTTACTTC ATTCTCACTG CAATCTATCA ACAACTAAAT 1130 AGATGGAAATACATCCAGCA GGTCTACCTG GCTAGCTTTT GGCTGGCAAT GAGCTCAACC 1190 ATGTACAATCCCATCATCTA CTGCTGTCTG AATAAAAGAT TTCGAGCTGG CTTCAAGAGA 1250 GCATTTCGCTGGTGTCCTTT CATCAAAGTT TCGAGCTATG ATGAGCTAGA GCTCAAGACC 1310 ACCAGGTTTCATCCAAACCG GCAAAGCAGT ATGTACACCG TGACCAGAAT GGAGTCCATG 1370 ACAGTCGTGTTTGACCCCAA CGATGCAGAC ACCACCAGGT CCAGTCGGAA GAAAAGAGCA 1430 ACGCCAAGAGACCCAAGTTT CAATGGCTGC TCTCGCAGGA ATTCCAAATC TGCCTCCGCC 1490 ACTTCAAGTTTCATAAGCTC ACCCTATACC TCTGTGGATG AATATTCTTA ATTCCATTTC 1550 CTGAGGTAAAAGATTAGTGT GAGACCATCA TGGTGCCAGT CTAGGACCCC ATTCTCCTAT 1610 TTATCAGTCCTGTCCTATAT ACCCTCTAGA AACAGAAAGC AATTTTTAGG CAGCTATGGT 1670 CAAATTGAGAAAGGTAGTGT ATAAATGTGA CAAAGACACT AATAACATGT TAGCCTCCAC 1730 CCAAAATAAAATGGGCTTTA AATTT 1755

or a degenerate variation thereof.

A third embodiment of this invention concerns systems for expressing allor part of the human neurokinin-3 receptor.

One class this third embodiment of the invention comprises:

A plasmid which comprises:

(a) a mammalian expression vector, such as pcDNAI/Neo, and

(b) a base sequence encoding human neurokinin-3 receptor protein.

Within this first class of the third embodiment the neurokinin-3receptor comprises the nucleotide sequence (SEQ ID NO:2:) ofcomplementary DNA as shown above.

A second class of this third embodiment of the invention concerns asystem for the transient expression of human neurokinin-3 receptor in amonkey kidney cell line (COS), the system comprised of a vector whichexpresses human neurokinin receptor (human NK3R) cDNA.

Within this second class of the third embodiment is the sub-classwherein the expression system includes:

A plasmid which comprises:

(a) a mammalian expression vector, such as pcDNAI/Neo, and

(b) a base sequence encoding human neurokinin-3 receptor protein.

A third class of this third embodiment of the invention concerns asystem for the expression of human neurokinin-3 receptor in a chinesehamster ovarian cell line (CHO), the. system comprising a vectorcomprising human neurokinin-3 receptor (human NK3R) cDNA.

Within this third class of the third embodiment is the sub-class whereinthe expression system includes:

A plasmid which comprises:

(a) a mammalian expression vector, such as pcNDAI/Neo and

(b) a base sequence encoding human neurokinin-3 receptor protein.

Within this sub-class the neurokinin-3 receptor expression systemcomprises the nucleotide sequence (SEQ ID NO:2:) of complementary DNA asshown above.

It is understood, and is readily apparent to those skilled in the artthat a wide variety of commonly used cell lines are suitable for use inthe present invention. Suitable cell lines derived from various speciesinclude, but are not limited to, cell lines of human, bovine, porcine,monkey, and rodent origin, or from yeast and bacterial strains.

A fourth embodiment of the invention concerns a method of using any ofthe above expression systems for determining the binding affinity of atest sample for human neurokinin-3 receptor.

In one class this fourth embodiment concerns a method of using a Chinesehamster ovarian cell line (CHO), the line transplanted with a plasmid,

which plasmid comprises:

(a) a mammalian expression vector, such as pcDNAI/Neo, and

(b) a base sequence encoding human neurokinin-3 receptor protein, themethod which comprises:

(1) expressing human neurokinin-3 receptor in the CHO cells;

(2) adding of a test sample to a solution containing ¹²⁵I-eledoisin andthe CHO cells;

(3) incubating the products of Step (2), the incubation being effectivefor competitive binding of the ¹²⁵I-eledoisin and said test sample tothe human neurokinin-3 receptor;

(4) separating the ¹²⁵I-eledoisin which is bound to the humanneurokinin-3 receptor from the ¹²⁵I-eledoisin which is not bound;

(5) measuring the amount of the ¹²⁵I-eledoisin which is bound to thehuman neurokinin-3 receptor.

In a second class this fourth embodiment concerns a method of using amonkey kidney cell line (COS), the line transplanted with a plasmid,.

which plasmid comprises:

(a) a mammalian expression vector, such as pcDNAI/Neo, and

(b) a base sequence encoding human neurokinin-3 receptor protein, themethod which comprises:

(1) expressing human neurokinin-3 receptor in the COS cells;

(2) adding of a test sample to a solution containing ¹²⁵I-eledoisin andthe COS cells;

(3) incubating the products of Step (2), the incubation being effectivefor competitive binding of the ¹²⁵I-eledoisin and said test sample tothe human neurokinin-3 receptor;

(4) separating the ¹²⁵I-eledoisin which is bound to the humanneurokinin-3 receptor from the ¹²⁵I-eledoisin which is not bound;

(5) measuring the amount of the ¹²⁵I-eledoisin which is bound to thehuman neurokinin-3 receptor.

In a third class this fourth embodiment concerns a method of using aChinese hamster ovarian cell line (CHO), the line transplanted with aplasmid,

which plasmid comprises:

(a) a mammalian expression vector, such as pcDNAI/Neo, and

(b) the base sequence encoding human neurokinin-3 receptor protein, themethod which comprises:

(1) expressing human neurokinin-3 receptor in the CHO cells;

(2) equilibrating the product of Step (1) with ³H-myoinositol;

(3) washing the product of Step (2);

(4) incubating the product of Step (3) with a test sample andneurokinin-B in the presence of aqueous LiCl, resulting in theproduction of ³H-inositol monophosphate;

(5) measuring the ³H-inositol monophosphate.

In overview, the present invention describes methods to isolate thehuman neurokinin-3 receptor (human NK3R) complementary DNA (cDNA)without prior knowledge of its protein sequence or gene sequence. Apolymerase chain reaction (PCR) technique was utilized for the isolationof human NK3R cDNA. In the approach, the regions of rat NK3R sequencethought to be similar to human NK3R were identified, oligonucleotideprimers corresponding to those region were designed, PCR amplificationwas carried out to obtain a partial clone of the NK3R cDNA from humancells, and its DNA sequence was determined. The full length cDNAencoding the human NK3R was obtained from human mRNA utilizing theprevious sequence information.

The complete sequence of the human NK3R CDNA was determined,.and itsencoded protein sequence was deduced. Among other things, such sequenceinformation is useful in the process of developing novel neurokinin Bantagonists.

Three heterologous expression systems were developed to express thecloned human NK3R cDNA. The Xenopus oocyte expression enables one todetermine the biological function of human NK3R . The COS (a monkeykidney cell line) expression can be used to measure the ligand bindingproperties of human NK3R. The CHO (a Chinese hamster ovarian cell line)stable expression is suitable for natural product screen to identifypotential therapeutic agents or other substances that bind toneurokinin-3 receptor or human NK3R. The cell line can also be used fordetermining the concentration of neurokinin B in human samples.

Assay protocols were developed to use the heterologously expressed humanNK3R for the. determination of the binding affinity and efficacy ofneurokinin B agonists/antagonists with therapeutic potential.

The following examples are given for the purpose of illustrating thepresent invention and shall not be construed as being limitations on thescope or spirit of the instant invention.

EXAMPLE 1

Isolation of human NK3R cDNA

To isolate the human NK3R cDNA in the absence of its sequenceinformation, we developed methods to obtain three separate butoverlapping cDNA clones in three steps. (i) We have adopted thehomologous cloning strategy (Ohara et al., Proc. Nat. Acad. Sci.,86:5673-5677 (1989)) to isolate cDNA clones encoding the central coreregion of human NK3R, with the assumption that the human NK3R sequenceis similar to the published sequence (Shigemoto et al., J. Biol. Chem.,265:623-628 (1990)) of rat NK3R in certain areas where appropriate PCRprimers can be designed. Degenerate primers corresponding to the ratsequence were used in PCR amplification (Mullis and Faloona, Meth.Enzymol., 155:335 (1987)) to obtain the cDNA encoding the centraltransmembrane core region of human NK3R from human mRNA. (ii) Afterdetermining the sequence of the core region in human NK3R, new primerscorresponding to the human sequence were designed and anchored PCRamplification (Frohman, et al., Proc. Nat. Acad. Sci., 85: 8998-9002(1988)) was performed using the human primer in the core region. ThecDNA encoding the N-terminal region of human NK3R was thus obtained fromhuman mRNA and its sequence was determined. (iii) An anchored PCRstrategy was also used to isolate the C-terminal region of human NK3R.To confirm the authenticity of the cDNA encoding human NK3R, anindependent PCR amplification was performed to obtain the full lengthcDNA in a single step using primers from the 5′ and 3′ untranslatedregions.

A cDNA clone encoding the central region of human NK3 receptor wasobtained from human brain mRNA by PCR using degenerate primers based onthe rat NK3 receptor sequence. The cDNA synthesis was initiated by theprimer “ca” (SEQ ID NO:3:)

GGATCCTCRTCRTAGCTGGANAC using reverse transcriptase from BRL(Gaithersburg, Md.). Primary PCR was performed at 50° C. annealingtemperature using the cDNA as template and primer “cb” (SEQ ID NO:4:)

TTTTGGATCCACTTGGATRAANGGRCA and primer “na” (SEQ ID NO:5:)

TTTTGGATCCTTCGTGCAGCCGTCCTGGCG

The following basic PCR conditions were used in all PCR experiments: 94°C. denaturation, 72° C. extension and 30 cycles. Secondary PCR wasperformed using the primary PCR product as template and the primer “cc”(SEQ ID NO:6:)

ATATGGATCCGACAGCAGCGAAATGCTCT and primer “nb” (SEQ ID NO:7:)

TTTTGAATTCTATGGCTTGGTGGTGGC under the same PCR conditions. A 900 bp cDNAfragment was obtained and sequenced, which was found to encode thecentral region of human NK3R.

A cDNA clone encoding the C-terminal region and 3′ untranslated regionof human NK3 receptor was obtained by anchored PCR using sense primersderived from the partial clone described above. The cDNA synthesis wasinitiated by the oligo-dT primer “notldt” (SEQ ID NO:8:)

TTTTGCGGCCGCTTTTTTTTTTTTTTTTT

It was followed by a tailing reaction using terminal deoxynucleotidetransferase (Promega, Madison, Wis.) to add a poly(A) tail to the 3′ endof the cDNA. Primary PCR was carried out using the cDNA as template andthe primers “notldt” and “s1068” (SEQ ID NO:9:)

AATTGGATCCTAGATGGAAATACATCCAGC at 55° C. annealing temperature.Secondary PCR was carried out using the primary PCR product as templateand the primers “notldt” and “s1106” (SEQ ID NO:10:)

AATTGGATCCTTGGCTGGCAATGAGCTCA under the same conditions. Tertiary PCRwas carried out using the secondary PCR product as template and theprimers “notldt” and “s1137” (SEQ ID NO:11:)

AATTGGATCCTCCCATCATCTACTGCTGTC under the same conditions. A 600 bp cDNAfragment was obtained and sequenced, which encodes the C-terminal regionof human NK3R and 3′ untranslated region.

A cDNA clone encoding the N-terminal region and 5′ untranslated regionof human NK3 receptor was obtained by anchored PCR using antisenseprimers derived from the partial clone encoding the central region ofhuman NK3R. The cDNA synthesis was initiated using the primers“a475”(SEQ ID NO:12:)

TGGCGAACACAGCTGTGATA and “a400” (SEQ ID NO:13:)

AGCGCGTAGATGAAATTGAC

A poly(A) tail was then added to the 3′ end of the cDNA. Primary PCR wasperformed using the cDNA as template and the primers “not1dt” and “a351”(SEQ ID NO:14:)

AATTGCGGCCGCCGGAGAAAGCCAGGTTCACA at 60° C. annealing temperature.Secondary PCR was performed using the primary PCR product as templateand the primers “not1dt” and “a332” (SEQ ID NO:15:)

AATTGCGGCCGCAGGAAGTAGTTGGTGACAGTC under the same conditions. A 600 bpcDNA fragment was obtained and sequenced, which encodes the N-terminalregion of human NK3R and 5′ untranslated region.

To confirm the authenticity of the human NK3R cDNA sequence, anindependent PCR was carried out to obtain the full length cDNA usingprimers based on the 5′ and 3′ untranslated regions. The cDNA wasinitiated using the primers “c1” (SEQ ID NO:16:)

AATTGCGGCCGCGACAGGACTGATAAATAGGAG and “c2” (SEQ ID NO:17:)

AATTGCGGCCGCCATGATGGTCTCACACTAATC

Primary PCR was performed using the cDNA as template and using theprimers “c2” and “s50” (SEQ ID NO:18:)

AAAGTGACCAGGAGGCAGAGA at 60° C. annealing temperature. Secondary PCR wasperformed using the primary PCR product as template and the primers “c3”(SEQ ID NO:19:)

AATTGCGGCCGCACCTCAGGAAATGGAATTAAG and “s71” (SEQ ID NO:20:)

AATTGGATCCAGAACTTCAGAGGAGTCTCG under the same conditions. A 1500 bp cDNAfragment was obtained and its sequence was consistent with the previouspartial clones.

EXAMPLE 2

Expression of the Cloned Human NK3R

Three expression systems were developed for the cloned human NK3R. Antransient expression in Xenopus oocytes resulted from microinjection ofin vitro transcribed mRNA from the cloned cDNA (Xenopus Laevis fromXENOPUS ONE, Ann Arbor, Mich.). This system allows the measurement ofbiological effect of NK3R activation upon ligand binding. Anothertransient expression in COS (a monkey kidney cell line, ATCC CRL 1651,ATCC Rockville Md.) resulted from the transfection of the cloned cDNAunder the control of viral promoter into mammalian cells (e.g., COS).The transfected cells are suitable for determination of the bindingaffinity of human NK3R for various ligands. Stable expression of humanNK3R in mammalian cells (e.g., CHO, a Chinese hamster ovarian cell line,ATCC CRL 9096, ATCC Rockville Md.) was achieved after integration of thetransfected cDNA into the chromosomes of the host cells. These stablecell lines will constituently express the cloned human NK3R and can bepropagated infinitely. Therefore, a stable expression system is veryuseful in large scale drug screening, and can be used to determine theconcentration of neurokinin-B related substances in biopsy samples ofpatients.

To express the cloned human NK3R, the full length cDNA of human NK3receptor was subcloned into the expression vector pcDNA-Neo (Invitrogen,San Diego, Calif.). Transient expression in COS cells was achieved byelectroporation using the IBI GeneZapper (IBI, New Haven, Conn.). Thetransfected cells were incubated in 10% fetal calf serum, 100 U/mlpenicillin-streptomycin, and 90% DMEM media (Gibco, Grand Island, N.Y.)in a 5% CO₂ incubator at 37° C. for three days before the binding assay.

To establish a stable cell line expressing the cloned human NK3R, thecDNA in the expression vector pcDNA-Neo was transfected into CHO cellsby electroporation. The transfected cells were incubated in theselection media (10% fetal calf serum, 100 U/ml penicillin-streptomycin,1/500 hypoxanthine-thymidine, 90% IMDM media (JRH Biosciences, Lenexa,KS), 0.7 mg/ml neomycin) in a 5% CO₂ incubator until colonies werevisible. Each colony was separated and propagated to maintain stablecell lines.

Both the COS expression and CHO expression allow the determination ofbinding affinity of various agonists and antagonists at the human NK3R.

The cloned human NK3R was expressed in Xenopus oocytes to demonstratethe biological function of human NK3 receptor as an activator of thesecond messenger inositol trisphosphate. The in vitro MRNA transcriptwas synthesized from the cDNA in pcDNA-Neo using T7 RNA polymerase(Stratagene, San Diego, Calif.) and injected into Xenopus oocytes. Theoocytes were incubated at 19° C. for two days beforeelectrophysiological assay.

EXAMPLE 3

Assays

The binding assay of human NK3R expressed in COS cells or CHO cells isbased on the use of ¹²⁵I-Bolton Hunter labeled eledoisin ( ¹²⁵I-BHE,from Du Pont, Boston, Mass.) (or ¹²⁵I-NKB) as a radioactively labeledligand which compete with unlabeled neurokinin peptides or any otherligand for binding to the human NK3R. Monolayer cell culture of COS orCHO was dissociated by the non-enzymatic solution (Specialty Media,Lavallette, N.J.) and resuspended in appropriate volume of the bindingbuffer (50 mM Tris pH 7.5, 5 mM MnCl₂, 150 mM NaCl, 0.04 mg/mlbacitracin, 0.004 mg/ml leupeptin, 0.2 mg/ml BSA, 0.01 mMphosphoramidon) such that 0.2 ml of the cell suspension would give riseto about 10,000 cpm of specific ¹²⁵I-BHE binding (approximately 50,000to 200,000 cells). In the binding assay, 0.2 ml of cells were added to atube containing 0.02 ml of 2.5 nM of ¹²⁵I-BHE and 0.02 ml of unlabeledtest compound. The tubes were incubated at 4° C. for 1 hour with gentleshaking. The bound radioactivity was separated from unboundradioactivity by GF/C filter (Brandel, Gaithersburg, Md.) which waspre-wetted with 0.1% polyethylenimine. The filter was washed with 3 mlof wash buffer (50 mM Tris pH 7.5, 5 mM MnCl₂, 150 mM NaCl) three timesand its radioactivity was determined by gamma counter.

The electrophysiological assay of human NK3R expressed in Xenopusoocytes was based on the fact that NK3R activates the phospholipase Cupon agonist binding, and phospholipase C in turn increases theintracellular calcium concentration through inositol trisphosphate (IP₃)and IP₃-gated calcium channel on intracellular membranes. The calciumincrease activates calcium-gated chloride channels on plasma membraneswhich gives rise to a chloride current measurable by two electrodevoltage clamp.

The oocyte was voltage-clamped at −80 mV by the model 8500 intracellularpreamp-clamp (Dagan, Minneapolis, Minn.). The recoding chamber wascontinuously perfused with recording buffer (96 mM NaCl, 2 mM KC1, 1.8mM CaCl₂, 5 mM HEPES, pH 7.4). Chloride current was elicited by applyingagonist (from 0.1 nM to 1000 nM) to the recording chamber. At leastthree oocytes were measured for each concentration. The antagonisticactivity of any potential NK3 antagonist can be assessed by determiningthe inhibition of neurokinin B response. Likewise, NK3 agonists can beidentified by their ability to stimulate a response in oocytes injectedwith NK3R mRNA but not in uninjected oocytes.

The activation of phospholipase C by the human NK3R may also be measuredin CHO cells by determining the accumulation of inositol monophosphatewhich is a degradation product of IP₃. CHO cells are seeded in 12-wellplate at 250,000 cells per well. After incubating in CHO media for 4days, cells are loaded with 0.025 mCi/ml of ³H-myoinositol by overnightincubation. The extracellular radioactivity is removed by washing withphosphate buffered saline. LiCl is added to the well at finalconcentration of 0.1 mM with or without antagonist, and continuedincubation at 37° C. for 15 min. Neurokinin B is added to the well atfinal concentration of 0.3 nM to activate the human NK3R. After 30 minof incubation at 37° C., the media is removed and 0.1 N HCl is added.Each well is sonicated at 4° C. and extracted with CHCl₃/methanol (1:1).The aqueous phase is applied to a 1 ml Dowex AG 1×8 ion exchange column.The column is washed with 0.1 N formic acid followed by 0.025 M ammoniumformate-0.1 N formic acid. The inositol monophosphate is eluted with 0.2M ammonium formate-0.1 N formic acid and quantitated by beta counter.

In addition to large scale drug screening using the stable CHO cell lineexpressing the cloned human NK3R, other alternative applications areobvious. For example, the stable cell line can be used in an assay todetermine the neurokinin B concentration in human samples.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be understood that the practice of the invention encompasses all ofthe casual variations, adaptations, modifications, deletions, oradditions of procedures and protocols described herein, as come withinthe scope of the following claims and its equivalents.

20 465 amino acids amino acid single linear protein not provided 1 MetAla Thr Leu Pro Ala Ala Glu Thr Trp Ile Asp Gly Gly Gly Gly 1 5 10 15Val Gly Ala Asp Ala Val Asn Leu Thr Ala Ser Leu Ala Ala Gly Ala 20 25 30Ala Thr Gly Ala Val Glu Thr Gly Trp Leu Gln Leu Leu Asp Gln Ala 35 40 45Gly Asn Leu Ser Ser Ser Pro Ser Ala Leu Gly Leu Pro Val Ala Ser 50 55 60Pro Ala Pro Ser Gln Pro Trp Ala Asn Leu Thr Asn Gln Phe Val Gln 65 70 7580 Pro Ser Trp Arg Ile Ala Leu Trp Ser Leu Ala Tyr Gly Val Val Val 85 9095 Ala Val Ala Val Leu Gly Asn Leu Ile Val Ile Trp Ile Ile Leu Ala 100105 110 His Lys Arg Met Arg Thr Val Thr Asn Tyr Phe Leu Val Asn Leu Ala115 120 125 Phe Ser Asp Ala Ser Met Ala Ala Phe Asn Thr Leu Val Asn PheIle 130 135 140 Tyr Ala Leu His Ser Glu Trp Tyr Phe Gly Ala Asn Tyr CysArg Phe 145 150 155 160 Gln Asn Phe Phe Pro Ile Thr Ala Val Phe Ala SerIle Tyr Ser Met 165 170 175 Thr Ala Ile Ala Val Asp Arg Tyr Met Ala IleIle Asp Pro Leu Lys 180 185 190 Pro Arg Leu Ser Ala Thr Ala Thr Lys IleVal Ile Gly Ser Ile Trp 195 200 205 Ile Leu Ala Phe Leu Leu Ala Phe ProGln Cys Leu Tyr Ser Lys Thr 210 215 220 Lys Val Met Pro Gly Arg Thr LeuCys Phe Val Gln Trp Pro Glu Gly 225 230 235 240 Pro Lys Gln His Phe ThrTyr His Ile Ile Val Ile Ile Leu Val Tyr 245 250 255 Cys Phe Pro Leu LeuIle Met Gly Ile Thr Tyr Thr Ile Val Gly Ile 260 265 270 Thr Leu Trp GlyGly Glu Ile Pro Gly Asp Thr Cys Asp Lys Tyr His 275 280 285 Glu Gln LeuLys Ala Lys Arg Lys Val Val Lys Met Met Ile Ile Val 290 295 300 Val MetThr Phe Ala Ile Cys Trp Leu Pro Tyr His Ile Tyr Phe Ile 305 310 315 320Leu Thr Ala Ile Tyr Gln Gln Leu Asn Arg Trp Lys Tyr Ile Gln Gln 325 330335 Val Tyr Leu Ala Ser Phe Trp Leu Ala Met Ser Ser Thr Met Tyr Asn 340345 350 Pro Ile Ile Tyr Cys Cys Leu Asn Lys Arg Phe Arg Ala Gly Phe Lys355 360 365 Arg Ala Phe Arg Trp Cys Pro Phe Ile Lys Val Ser Ser Tyr AspGlu 370 375 380 Leu Glu Leu Lys Thr Thr Arg Phe His Pro Asn Arg Gln SerSer Met 385 390 395 400 Tyr Thr Val Thr Arg Met Glu Ser Met Thr Val ValPhe Asp Pro Asn 405 410 415 Asp Ala Asp Thr Thr Arg Ser Ser Arg Lys LysArg Ala Thr Pro Arg 420 425 430 Asp Pro Ser Phe Asn Gly Cys Ser Arg ArgAsn Ser Lys Ser Ala Ser 435 440 445 Ala Thr Ser Ser Phe Ile Ser Ser ProTyr Thr Ser Val Asp Glu Tyr 450 455 460 Ser 465 1755 base pairs nucleicacid single linear cDNA not provided 2 CTATTGCAGT ATCTTTCAGC TTCCAGTCTTATCTGAAGAC CCCGGCACCA AAGTGACCAG 60 GAGGCAGAGA AGAACTTCAG AGGAGTCTCGTCTTGGGCTG CCCGTGGGTG AGTGGGAGGG 120 TCCGGGACTG CAGACCGGTG GCGATGGCCACTCTCCCAGC AGCAGAAACC TGGATAGACG 180 GGGGTGGAGG CGTGGGTGCA GACGCCGTGAACCTGACCGC CTCGCTAGCT GCCGGGGCGG 240 CCACGGGGGC AGTTGAGACT GGGTGGCTGCAACTGCTGGA CCAAGCTGGC AACCTCTCCT 300 CCTCCCCTTC CGCGCTGGGA CTGCCTGTGGCTTCCCCCGC GCCCTCCCAG CCCTGGGCCA 360 ACCTCACCAA CCAGTTCGTG CAGCCGTCCTGGCGCATCGC GCTCTGGTCC CTGGCGTATG 420 GTGTGGTGGT GGCAGTGGCA GTTTTGGGAAATCTCATCGT CATCTGGATC ATCCTGGCCC 480 ACAAGCGCAT GAGGACTGTC ACCAACTACTTCCTTGTGAA CCTGGCTTTC TCCGACGCCT 540 CCATGGCCGC CTTCAACACG TTGGTCAATTTCATCTACGC GCTTCATAGC GAGTGGTACT 600 TTGGCGCCAA CTACTGCCGC TTCCAGAACTTCTTTCCTAT CACAGCTGTG TTCGCCAGCA 660 TCTACTCCAT GACGGCCATT GCGGTGGACAGGTATATGGC TATTATTGAT CCCTTGAAAC 720 CCAGACTGTC TGCTACAGCA ACCAAGATTGTCATTGGAAG TATTTGGATT CTAGCATTTC 780 TACTTGCCTT CCCTCAGTGT CTTTATTCCAAAACCAAAGT CATGCCAGGC CGTACTCTCT 840 GCTTTGTGCA ATGGCCAGAA GGTCCCAAACAACATTTCAC TTACCATATT ATCGTCATTA 900 TACTGGTGTA CTGTTTCCCA TTGCTCATCATGGGTATTAC ATACACCATT 950 GTTGGAATTA CTCTCTGGGG AGGAGAAATC CCAGGAGATACCTGTGACAA GTATCATGAG 1010 CAGCTAAAGG CCAAAAGAAA GGTTGTCAAA ATGATGATTATTGTTGTCAT GACATTTGCT 1070 ATCTGCTGGC TGCCCTATCA TATTTACTTC ATTCTCACTGCAATCTATCA ACAACTAAAT 1130 AGATGGAAAT ACATCCAGCA GGTCTACCTG GCTAGCTTTTGGCTGGCAAT GAGCTCAACC 1190 ATGTACAATC CCATCATCTA CTGCTGTCTG AATAAAAGATTTCGAGCTGG CTTCAAGAGA 1250 GCATTTCGCT GGTGTCCTTT CATCAAAGTT TCCAGCTATGATGAGCTAGA GCTCAAGACC 1310 ACCAGGTTTC ATCCAAACCG GCAAAGCAGT ATGTACACCGTGACCAGAAT GGAGTCCATG 1370 ACAGTCGTGT TTGACCCCAA CGATGCAGAC ACCACCAGGTCCAGTCGGAA GAAAAGAGCA 1430 ACGCCAAGAG ACCCAAGTTT CAATGGCTGC TCTCGCAGGAATTCCAAATC TGCCTCCGCC 1490 ACTTCAAGTT TCATAAGCTC ACCCTATACC TCTGTGGATGAATATTCTTA ATTCCATTTC 1550 CTGAGGTAAA AGATTAGTGT GAGACCATCA TGGTGCCAGTCTAGGACCCC ATTCTCCTAT 1610 TTATCAGTCC TGTCCTATAT ACCCTCTAGA AACAGAAAGCAATTTTTAGG CAGCTATGGT 1670 CAAATTGAGA AAGGTAGTGT ATAAATGTGA CAAAGACACTAATAACATGT TAGCCTCCAC 1730 CCAAAATAAA ATGGGCTTTA AATTT 1755 23 basepairs nucleic acid single linear cDNA not provided 3 GGATCCTCRTCRTAGCTGGA NAC 23 27 base pairs nucleic acid single linear cDNA notprovided 4 TTTTGGATCC ACTTGGATRA ANGGRCA 27 30 base pairs nucleic acidsingle linear cDNA not provided 5 TTTTGGATCC TTCGTGCAGC CGTCCTGGCG 30 29base pairs nucleic acid single linear cDNA not provided 6 ATATGGATCCGACAGCAGCG AAATGCTCT 29 27 base pairs nucleic acid single linear cDNAnot provided 7 TTTTGAATTC TATGGCTTGG TGGTGGC 27 29 base pairs nucleicacid single linear cDNA not provided 8 TTTTGCGGCC GCTTTTTTTT TTTTTTTTT29 30 base pairs nucleic acid single linear cDNA not provided 9AATTGGATCC TAGATGGAAA TACATCCAGC 30 29 base pairs nucleic acid singlelinear cDNA not provided 10 AATTGGATCC TTGGCTGGCA ATGAGCTCA 29 30 basepairs nucleic acid single linear cDNA not provided 11 AATTGGATCCTCCCATCATC TACTGCTGTC 30 20 base pairs nucleic acid single linear cDNAnot provided 12 TGGCGAACAC AGCTGTGATA 20 20 base pairs nucleic acidsingle linear cDNA not provided 13 AGCGCGTAGA TGAAATTGAC 20 32 basepairs nucleic acid single linear cDNA not provided 14 AATTGCGGCCGCCGGAGAAA GCCAGGTTCA CA 32 33 base pairs nucleic acid single linearcDNA not provided 15 AATTGCGGCC GCAGGAAGTA GTTGGTGACA GTC 33 33 basepairs nucleic acid single linear cDNA not provided 16 AATTGCGGCCGCGACAGGAC TGATAAATAG GAG 33 33 base pairs nucleic acid single linearcDNA not provided 17 AATTGCGGCC GCCATGATGG TCTCACACTA ATC 33 21 basepairs nucleic acid single linear cDNA not provided 18 AAAGTGACCAGGAGGCAGAG A 21 33 base pairs nucleic acid single linear cDNA notprovided 19 AATTGCGGCC GCACCTCAGG AAATGGAATT AAG 33 30 base pairsnucleic acid single linear cDNA not provided 20 AATTGGATCC AGAACTTCAGAGGAGTCTCG 30

What is claimed is:
 1. A human neurokinin-3 receptor protein comprisingthe amino acid sequence (SEQ ID NO:1:) which is: Met Ala Thr Leu Pro AlaAla Glu 1               5 Thr Trp Ile Asp Gly Gly Gly Gly    10                  15 Val Gly Ala Asp Ala Val Asn Leu            20 Thr Ala Ser Leu Ala Ala Gly Ala 25                  30Ala Thr Gly Ala Val Glu Thr Gly         35                  40 Trp LeuGln Leu Leu Asp Gln Ala                 45 Gly Asn Leu Ser Ser Ser ProSer     50                  55 Ala Leu Gly Leu Pro Val Ala Ser            60 Pro Ala Pro Ser Gln Pro Trp Ala 65                  70Asn Leu Thr Asn Gln Phe Val Gln         75                  80 Pro SerTrp Arg Ile Ala Leu Trp                 85 Ser Leu Ala Tyr Gly Val ValVal     90                  95 Ala Val Ala Val Leu Gly Asn Leu            100 Ile Val Ile Trp Ile Ile Leu Ala 105                 110His Lys Arg Met Arg Thr Val Thr         115                 120 Asn TyrPhe Leu Val Asn Leu Ala                 125 Phe Ser Asp Ala Ser Met AlaAla     130                 135 Phe Asn Thr Leu Val Asn Phe Ile            140 Tyr Ala Leu His Ser Glu Trp Tyr 145                 150Phe Gly Ala Asn Tyr Cys Arg Phe         155                 160 Gln AsnPhe Phe Pro Ile Thr Ala                 165 Val Phe Ala Ser Ile Tyr SerMet     170                 175 Thr Ala Ile Ala Val Asp Arg Tyr            180 Met Ala Ile Ile Asp Pro Leu Lys 185                 190Pro Arg Leu Ser Ala Thr Ala Thr         195                 200 Lys IleVal Ile Gly Ser Ile Trp                 205 Ile Leu Ala Phe Leu Leu AlaPhe     210                 215 Pro Gln Cys Leu Tyr Ser Lys Thr            220 Lys Val Met Pro Gly Arg Thr Leu 225                 230Cys Phe Val Gln Trp Pro Glu Gly         235                 240 Pro LysGln His Phe Thr Tyr His                 245 Ile Ile Val Ile Ile Leu ValTyr     250                 255 Cys Phe Pro Leu Leu Ile Met Gly            260 Ile Thr Tyr Thr Ile Val Gly Ile 265                 270Thr Leu Trp Gly Gly Glu Ile Pro         275                 280 Gly AspThr Cys Asp Lys Tyr His                 285 Glu Gln Leu Lys Ala Lys ArgLys     290                 295 Val Val Lys Met Met Ile Ile Val            300 Val Met Thr Phe Ala Ile Cys Trp 305                 310Leu Pro Tyr His Ile Tyr Phe Ile         315                 320 Leu ThrAla Ile Tyr Gln Gln Leu                 325 Asn Arg Trp Lys Tyr Ile GlnGln     330                 335 Val Tyr Leu Ala Ser Phe Trp Leu            340 Ala Met Ser Ser Thr Met Tyr Asn 345                 350Pro Ile Ile Tyr Cys Cys Leu Asn         355                 360 Lys ArgPhe Arg Ala Gly Phe Lys                 365 Arg Ala Phe Arg Trp Cys ProPhe     370                 375 Ile Lys Val Ser Ser Tyr Asp Glu            380 Leu Glu Leu Lys Thr Thr Arg Phe 385                 390His Pro Asn Arg Gln Ser Ser Met         395                 400 Tyr ThrVal Thr Arg Met Glu Ser                 405 Met Thr Val Val Phe Asp ProAsn     410                 415 Asp Ala Asp Thr Thr Arg Ser Ser            420 Arg Lys Lys Arg Ala Thr Pro Arg 425                 430Asp Pro Ser Phe Asn Gly Cys Ser         435                 440 Arg ArgAsn Ser Lys Ser Ala Ser                 445 Ala Thr Ser Ser Phe Ile SerSer     450                 455 Pro Tyr Thr Ser Val Asp Glu Tyr            460 Ser 465

the receptor protein being free of other human receptor proteins.
 2. ADNA molecule en oding the human neurokinin-3 receptor of claim 1, theDNA molecule being free of other human DNA molecules.
 3. A DNA moleculeencoding human neurokinin-3 receptor comprising the nucleotide sequence(SEQ ID NO:2:) which is: CTATTGCAGT ATCTTTCAGC TTCCAGTCTT ATCTGAAGACCCCGGCACCA AAGTGACCAG 60 GAGGCAGAGA AGAACTTCAG AGGAGTCTCG TCTTGGGCTGCCCGTGGGTG AGTGGGAGGG 120 TCCGGGACTG CAGACCGGTG GCGATGGCCA CTCTCCCAGCAGCAGAAACC TGGATAGACG 180 GGGGTGGAGG CGTGGGTGCA GACGCCGTGA ACCTGACCGCCTCGCTAGCT GCCGGGGCGG 240 CCACGGGGGC AGTTGAGACT GGGTGGCTGC AACTGCTGGACCAAGCTGGC AACCTCTCCT 300 CCTCCCCTTC CGCGCTGGGA CTGCCTGTGG CTTCCCCCGCGCCCTCCCAG CCCTGGGCCA 360 ACCTCACCAA CCAGTTCGTG CAGCCGTCCT GGCGCATCGCGCTCTGGTCC CTGGCGTATG 420 GTGTGGTGGT GGCAGTGGCA GTTTTGGGAA ATCTCATCGTCATCTGGATC ATCCTGGCCC 480 ACAAGCGCAT GAGGACTGTC ACCAACTACT TCCTTGTGAACCTGGCTTTC TCCGACGCCT 540 CCATGGCCGC CTTCAACACG TTGGTCAATT TCATCTACGCGCTTCATAGC GAGTGGTACT 600 TTGGCGCCAA CTACTGCCGC TTCCAGAACT TCTTTCCTATCACAGCTGTG TTCGCCAGCA 660 TCTACTCCAT GACGGCCATT GCGGTGGACA GGTATATGGCTATTATTGAT CCCTTGAAAC 720 CCAGACTGTC TGCTACAGCA ACCAAGATTG TCATTGGAAGTATTTGGATT CTAGCATTTC 780 TACTTGCCTT CCCTCAGTGT CTTTATTCCA AAACCAAAGTCATGCCAGGC CGTACTCTCT 840 GCTTTGTGCA ATGGCCAGAA GGTCCCAAAC AACATTTCACTTACCATATT ATCGTCATTA 900 TACTGGTGTA CTGTTTCCCA TTGCTCATCA TGGGTATTACATACACCATT 950 GTTGGAATTA CTCTCTGGGG AGGAGAAATC CCAGGAGATA CCTGTGACAAGTATCATGAG 1010 CAGCTAAAGG CCAAAAGAAA GGTTGTCAAA ATGATGATTA TTGTTGTCATGACATTTGCT 1070 ATCTGCTGGC TGCCCTATCA TATTTACTTC ATTCTCACTG CAATCTATCAACAACTAAAT 1130 AGATGGAAAT ACATCCAGCA GGTCTACCTG GCTAGCTTTT GGCTGGCAATGAGCTCAACC 1190 ATGTACAATC CCATCATCTA CTGCTGTCTG AATAAAAGAT TTCGAGCTGGCTTCAAGAGA 1250 GCATTTCGCT GGTGTCCTTT CATCAAAGTT TCCAGCTATG ATGAGCTAGAGCTCAAGACC 1310 ACCAGGTTTC ATCCAAACCG GCAAAGCAGT ATGTACACCG TGACCAGAATGGAGTCCATG 1370 ACAGTCGTGT TTGACCCCAA CGATGCAGAC ACCACCAGGT CCAGTCGGAAGAAAAGAGCA 1430 ACGCCAAGAG ACCCAAGTTT CAATGGCTGC TCTCGCAGGA ATTCCAAATCTGCCTCCGCC 1490 ACTTCAAGTT TCATAAGCTC ACCCTATACC TCTGTGGATG AATATTCTTAATTCCATTTC 1550 CTGAGGTAAA AGATTAGTGT GAGACCATCA TGGTGCCAGT CTAGGACCCCATTCTCCTAT 1610 TTATCAGTCC TGTCCTATAT ACCCTCTAGA AACAGAAAGC AATTTTTAGGCAGCTATGGT 1670 CAAATTGAGA AAGGTAGTGT ATAAATGTGA CAAAGACACT AATAACATGTTAGCCTCCAC 1730 CCAAAATAAA ATGGGCTTTA AATTT 1755

the DNA molecule being free of other human DNA molecules.
 4. A plasmidwhich comprises: (a) a mammalian expression vector, and (b) a nucleotidesequence encoding human neurokinin-3 receptor protein, wherein thenucleotide sequence (SEQ ID NO:2:) comprises: CTATTGCAGT ATCTTTCAGCTTCCAGTCTT ATCTGAAGAC CCCGGCACCA AAGTGACCAG 60 GAGGCAGAGA AGAACTTCAGAGGAGTCTCG TCTTGGGCTG CCCGTGGGTG AGTGGGAGGG 120 TCCGGGACTG CAGACCGGTGGCGATGGCCA CTCTCCCAGC AGCAGAAACC TGGATAGACG 180 GGGGTGGAGG CGTGGGTGCAGACGCCGTGA ACCTGACCGC CTCGCTAGCT GCCGGGGCGG 240 CCACGGGGGC AGTTGAGACTGGGTGGCTGC AACTGCTGGA CCAAGCTGGC AACCTCTCCT 300 CCTCCCCTTC CGCGCTGGGACTGCCTGTGG CTTCCCCCGC GCCCTCCCAG CCCTGGGCCA 360 ACCTCACCAA CCAGTTCGTGCAGCCGTCCT GGCGCATCGC GCTCTGGTCC CTGGCGTATG 420 GTGTGGTGGT GGCAGTGGCAGTTTTGGGAA ATCTCATCGT CATCTGGATC ATCCTGGCCC 480 ACAAGCGCAT GAGGACTGTCACCAACTACT TCCTTGTGAA CCTGGCTTTC TCCGACGCCT 540 CCATGGCCGC CTTCAACACGTTGGTCAATT TCATCTACGC GCTTCATAGC GAGTGGTACT 600 TTGGCGCCAA CTACTGCCGCTTCCAGAACT TCTTTCCTAT CACAGCTGTG TTCGCCAGCA 660 TCTACTCCAT GACGGCCATTGCGGTGGACA GGTATATGGC TATTATTGAT CCCTTGAAAC 720 CCAGACTGTC TGCTACAGCAACCAAGATTG TCATTGGAAG TATTTGGATT CTAGCATTTC 780 TACTTGCCTT CCCTCAGTGTCTTTATTCCA AAACCAAAGT CATGCCAGGC CGTACTCTCT 840 GCTTTGTGCA ATGGCCAGAAGGTCCCAAAC AACATTTCAC TTACCATATT ATCGTCATTA 900 TACTGGTGTA CTGTTTCCCATTGCTCATCA TGGGTATTAC ATACACCATT 950 GTTGGAATTA CTCTCTGGGG AGGAGAAATCCCAGGAGATA CCTGTGACAA GTATCATGAG 1010 CAGCTAAAGG CCAAAAGAAA GGTTGTCAAAATGATGATTA TTGTTGTCAT GACATTTGCT 1070 ATCTGCTGGC TGCCCTATCA TATTTACTTCATTCTCACTG CAATCTATCA ACAACTAAAT 1130 AGATGGAAAT ACATCCAGCA GGTCTACCTGGCTAGCTTTT GGCTGGCAAT GAGCTCAACC 1190 ATGTACAATC CCATCATCTA CTGCTGTCTGAATAAAAGAT TTCGAGCTGG CTTCAAGAGA 1250 GCATTTCGCT GGTGTCCTTT CATCAAAGTTTCCAGCTATG ATGAGCTAGA GCTCAAGACC 1310 ACCAGGTTTC ATCCAAACCG GCAAAGCAGTATGTACACCG TGACCAGAAT GGAGTCCATG 1370 ACAGTCGTGT TTGACCCCAA CGATGCAGACACCACCAGGT CCAGTCGGAA GAAAAGAGCA 1430 ACGCCAAGAG ACCCAAGTTT CAATGGCTGCTCTCGCAGGA ATTCCAAATC TGCCTCCGCC 1490 ACTTCAAGTT TCATAAGCTC ACCCTATACCTCTGTGGATG AATATTCTTA ATTCCATTTC 1550 CTGAGGTAAA AGATTAGTGT GAGACCATCATGGTGCCAGT CTAGGACCCC ATTCTCCTAT 1610 TTATCAGTCC TGTCCTATAT ACCCTCTAGAAACAGAAAGC AATTTTTAGG CAGCTATGGT 1670 CAAATTGAGA AAGGTAGTGT ATAAATGTGACAAAGACACT AATAACATGT TAGCCTCCAC 1730 CCAAAATAAA ATGGGCTTTA AATTT
 1755.


5. A Chinese hamster ovarian cell line, the cell line transfected withthe plasmid of claim
 4. 6. A monkey kidney cell line, the cell linetransfected with the plasmid of claim 4.